Expression process

ABSTRACT

A process for the production of a target polypeptide is provided. The process comprises expression of an expression vector for expressing a target polypeptide in a host cell, preferably a mammalian cell, the expression vector comprising an expression cassette comprising a polynucleotide encoding a recombinant polypeptide operably linked to a fibronectin secretion leader sequence; and recovering the target polypeptide.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled “SequenceListing.txt” created onor about Oct. 16, 2015, with a file size of about 2 kb contains thesequence listing for this application and is hereby incorporated byreference in its entirety.

The present invention concerns a process for the expression ofrecombinant polypeptides, and in particular the secretion of recombinantpolypeptides.

It is of significant benefit in recombinant polypeptide production ifthe polypeptide of interest can be exported from the cell in which it isexpressed. Expression systems are therefore advantageously designed toenable such export, or secretion. Secretion of the recombinantpolypeptide from the host cell commonly involves use of signal peptides,which are found on the majority of eukaryotic and prokaryotic proteinsthat are destined for export from the cytoplasm. Secretion leadersemployed in such expression systems are typically native to theexpression host, for example, the PhoA, MalB and OmpA signal peptides ofEscherichia coli have been used extensively to secrete polypeptides tothe periplasm of that organism.

U.S. Pat. No. 7,071,172 describes the use of fibronectin secretionleaders in AAV-based delivery vectors for use in gene therapy.

According to a first aspect of the present invention, there is provideda process for the production of a target polypeptide which comprises:

a) expressing an expression vector for expressing a target polypeptidein a host cell, the expression vector comprising an expression cassettecomprising a polynucleotide encoding a recombinant polypeptide operablylinked to a fibronectin secretion leader sequence or a functionalequivalent thereof; and

b) recovering the target polypeptide.

Fibronectin secretion leaders that can be employed in the presentinvention include mammalian and reptilian fibronectin secretion leaders.Examples of reptilian fibronectin secretion leaders include Xenopuslaevis fibronectin secretion leaders. Examples of mammalian fibronectinsecretion leaders include human, rat, murine, bovine, porcine, canine,feline and Chinese hamster fibronectin secretion leaders, and functionalequivalents thereof, such as human fibronectin secretion leader havingthe sequence MLRGPGPGLLLLAVQCLGTAVPSTGA (SEQ ID No. 1). In certainembodiments, the Chinese hamster fibronectin secretion leader having theamino acid sequence MLRGPGPGLLLAVLCLGTAVRCTEA (SEQ ID No. 2) andfunctional equivalents thereof is preferred.

A functional equivalent to a secretion leader is one that shares 70% orgreater identity with an amino acid sequence, preferably 75% or greateridentity, more preferably 80% or greater identity and most preferably90% or greater identity, such as 95% identity or more, and which retainsthe ability to secrete the recombinant polypeptide. In some embodiments,the functionally equivalent secretion leader differs by a single aminoacid, by any of addition, deletion or replacement.

In many embodiments, polynucleotide sequences which are operably linkedare contiguous and, in the case of a secretion leader, contiguous and inthe same reading frame.

Preferably, the linkage between the polynucleotide encoding thefibronectin secretion leader sequence and the polynucleotide encodingthe target polypeptide is such that the secretion leader is attached tothe N-terminal of the recombinant polypeptide. In certain embodiments,the recombinant polypeptide comprises an N-terminal tag, the linkagebetween the secretion leader sequence and the polynucleotide encodingthe recombinant polypeptide being such that the secretion leader isattached to the tag, preferably to the N-terminus of the tag.

The polynucleotide encoding the fibronectin secretion leader sequence ispreferably attached at the 5′ end of the polynucleotide encoding thetarget polypeptide and preferably has the sequenceATGCTGAGAGGCCCTGGACCTGGACTGCTGCTGCTGGCTGTGCAGTGTCTGGGAACCGCCGTGCCTTCTACCGGCGCC(SEQ ID No. 3) orATGCTCAGGGGTCCGGGACCCGGGCTGCTGCTGGCCGTCCTGTGCCTGGGGACAGCGGTGCGCTGTACCGAAGCC(SEQ ID No. 4).

The vectors of the present invention comprise a promoter operably linkedto the expression cassette for the secretion leader and recombinantpolypeptides.

Promoters which may be employed in the vectors according to the presentinvention are selected according to the host cell in which theexpression cassette is to be expressed.

Promoters that can be employed in prokaryotic host cells include phagepolymerase-promoters, such as single T7 promoter regions, includingthose disclosed by Studier and Moffat, J. Mol. Biol. 189:113-130 (1986),incorporated herein by reference, especially a T7 gene 10 promoterregion and host polymerase promoters, especially E coli polymerasepromoters, such as T7A1, T7A2, T7A3, λpL, λpR, lac, lacUV5, trp, tac,trc, phoA and rrnB.

When a T7 RNA-polymerase dependent promoter region is employed, it willbe recognised that a source of T7 RNA polymerase is required, which isprovided by methods known in the art, and commonly by inserting a λDE3prophage expressing the required phage polymerase into the host strainto create lysogenic host strains. The T7 RNA polymerase can also bedelivered to the cell by infection with a specialised λ transducingphage that carries the gene for the T7 RNA polymerase.

Promoters that can be employed in yeast host cell include gal promotersand AOX promoters, such as AOX1 and AOX2, GAP (glyceraldehyde3-phosphate dehydrogenase), FLP (formaldehyde dehydrogenase) and GAL1and GAL10.

Promoters that can be employed in mammalian host cells may be endogenousor exogenous to the host cells. Suitable promoters include viralpromoters such as CMV, SV40 promoter, and RSR-LTR. Promoters fromhousekeeping genes such as hEF1a and murine phosphoglycerate kinase(mPGK) may also be utilised. In some embodiments, preferred promotersare human CMV and rat CMV. The promoters may be the same or different ifmore than one polypeptide is being expressed (eg MAb HC and LCpolypeptides). The promoter may be employed in combination with anenhancer sequence, such as the major immediate early enhancer of acytomegalovirus, especially human cytomegalovirus.

The expression vector may be integrated into the host cell genome orcomprised within an extrachromosomal element such as a plasmid.

The expression vector typically also comprises a selectable markerappropriate to the host cell in which the vector is to be expressed.Selectable markers for use in prokaryotic host cells include antibioticresistance markers, such as tetracycline or kanamycin resistancemarkers. Selectable markers for use in yeast hosts include antibioticresistance markers, such as Zeocin, puromycin, neocin and hygromycinresistance. Selectable markers for mammalian cells, and especially forChinese hamster ovary cells include glutamine synthetase anddihydrofolate reductase marker systems.

The vectors employed comprise features conventional in the artappropriate for expression in the appropriate host cell. Prokaryoticexpression vectors typically comprise an origin of replication,restriction enzyme sites, a transcription terminator, and a plasmidstability locus, such as a cer stability sequence. Yeast expressionvectors typically comprise promoter, transcription terminator, selectionmarker, and if replicating, an origin of replication. Mammalianexpression vectors typically comprise a polyadenylation sequence, suchas human betaglobin polyA sequence, bovine growth hormone polyA sequenceand SV40 early or late poly A sequences.

The expression vector of the present invention can be employed toexpress recombinant polypeptides, especially proteins in host cells.Prokaryote and especially eukaryote host cells can be employed. Examplesof prokaryotic cells include bacterial cells, for example gram-negativebacterial cells, including E. coli, Salmonella typhimurium, Serratiamarsescens, Pseudomonas putida and Pseudomonas aeruginosa, andgram-positive bacterial cells including Bacillus subtilis. Preferredprokaryote host cells are bacteria, particularly enterobacteriacae,preferably E coli, and especially B or K12 strains thereof.

Examples of eukaryote host cells which can be employed include yeast,mammalian and insect cells. Yeast host cells include in particularSaccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha.

Preferred host cells are mammalian cells, such as baby hamster kidneycells, human embryonic kidney cell lines, for example HEK 293 cells,human retina-derived cell lines, for example PER.C6 cells, and murinelymphoid cell lines, for example NS0 and SP2 cells. and most preferablyChinese hamster ovary cells, and in particular CHOK1, DG44, DUXKB11 andCHO pro3-cells.

The expression vector of the present invention is commonly employed inthe form of a plasmid. The plasmids may be autonomously replicatingplasmids or integrative plasmids.

In certain highly preferred embodiments of the present invention, thefibronectin secretion leader is selected to correspond to the host cellemployed. For example, human fibronectin is employed in human-derivedcells, rat fibronectin is employed in rat cells, and particularlyChinese hamster fibronectin is employed in Chinese hamster ovary cells.

Polypeptides which can be expressed by the process of the presentinvention include therapeutic proteins and peptides, includingcytokines, growth factors, antibodies, antibody fragments,immunoglobulin like polypeptides, enzyme, vaccines, peptide hormones,chemokines, receptors, receptor fragments, kinases, phosphatases,isomerases, hydrolyases, transcription factors and fusion polypeptides.

Antibodies which can be expressed include monoclonal antibodies,polyclonal antibodies and antibody fragments having biological activity,including multivalent and/or multispecific forms of any of theforegoing.

Naturally occurring antibodies typically comprise four polypeptidechains, two identical heavy (H) chains and two identical light (L)chains inter-connected by disulfide bonds. Each heavy chain comprises avariable region (V_(H)) and a constant region (C_(H)), the C_(H) regioncomprising in its native form three domains, C_(H)1, C_(H)2 and C_(H)3.Each light chain comprises a variable region (V_(L)) and a constantregion comprising one domain, C_(L).

The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Antibody fragments which can be expressed comprise a portion of anintact antibody, said portion having a desired biological activity.Antibody fragments generally include at least one antigen binding site.Examples of antibody fragments include: (i) Fab fragments having V_(L),C_(L), V_(H) and C_(H)1 domains; (ii) Fab derivatives, such as a Fab′fragment having one or more cysteine residues at the C-terminus of theC_(H)1 domain, that can form bivalent fragments by disulfide bridgingbetween two Fab derivatives; (iii) Fd fragment having V_(H) and C_(H)1domains; (iv) Fd derivatives, such as Fd derivatives having one or morecysteine residues at the C-terminus of the C_(H)1 domain; (v) Fvfragments having the V_(L) and V_(H) domains of a single arm of anantibody; (vi) single chain antibody molecules such as single chain Fv(scFv) antibodies in which the V_(L) and V_(H) domains are covalentlylinked; (vii) V_(H) or V_(L) domain polypeptide without constant regiondomains linked to another variable domain (a V_(H) or V_(L) domainpolypeptide) that is with or without constant region domains, (e.g.,V_(H)-V_(H), V_(H)-V_(L), or V_(L)-V_(L)) (viii) domain antibodyfragments, such as fragments consisting of a V_(H) domain, or a V_(L)domain, and antigen-binding fragments of either V_(H) or V_(L) domains,such as isolated CDR regions; (ix) so-called “diabodies” comprising twoantigen binding sites, for example a heavy chain variable domain (V_(H))connected to a light chain variable domain (V_(L)), in the samepolypeptide chain; and (x) so-called linear antibodies comprising a pairof tandem Fd segments which, together with complementary light chainpolypeptides, form a pair of antigen binding regions.

Preferred antibody fragments that can be prepared are mammalian singlevariable domain antibodies, being an antibody fragment comprising afolded polypeptide domain which comprises sequences characteristic ofimmunoglobulin variable domains and which specifically binds an antigen(i.e., dissociation constant of 500 nM or less, such as 400 nM or less,preferably 250 nM or less, and most preferably 100 nM or less), andwhich binds antigen as a single variable domain; that is, without anycomplementary variable domain. Single variable domain antibodies includecomplete antibody variable domains as well as modified variable domains,for example in which one or more loops have been replaced by sequenceswhich are not characteristic of antibody variable domains or antibodyvariable domains which have been truncated or comprise N- or C-terminalextensions, as well as folded fragments of variable domains. Preferredsingle variable domains which can be prepared are selected from thegroup of V_(H) and V_(L), including V_(kappa) and V_(lambda). Mostpreferably the single variable domains are human or camelid domains,including humanised camelid domains.

Where the target polypeptide comprises two or more chains to besecreted, particularly where the target polypeptide is an antibody or afragment antibody comprising two or more chains, at least one, andpreferably each, of the chains is attached to a fibronectin secretionleader, and polynucleotides encoding such polypeptides are designedaccordingly. The fibronectin secretion leaders employed may be the sameor different. The polynucleotides encoding the two or more chains may becomprised within the same expression cassette, but are preferablycomprised in different expression cassettes. Where different expressioncassettes are employed, the expression cassettes may be located ondifferent vectors, but are preferably on the same vector. Promotersemployed may be the same or different.

The expression system is expressed by methods well known in the art forthe cells employed. Preferred expression methods include culturing thehost cells in growth medium, and then recovering the expressedpolypeptide. The term “growth medium” refers to a nutrient medium usedfor growing the host cells. In many embodiments, a nutrient solution isemployed. Suitable growth media for given host cells and methods ofrecovering polypeptides are well known in the art.

In many embodiments, the polypeptide recovery comprises one or more offiltration, centrifugation, diafiltration, ion-exchange chromatography,affinity chromatography, such as Protein A affinity chromatography,Hydrophobic Interaction Chromatography (HIC), Gel Filtration and HPLC.

According to a preferred aspect of the present invention there isprovided a process for the production of a target polypeptide whichcomprises:

(a) transfection or transformation of a host cell with an expressionvector for expressing a target polypeptide in a host cell, theexpression vector comprising an expression cassette comprising apolynucleotide encoding the target polypeptide operably linked to afibronectin secretion leader sequence or a functional equivalentthereof;

(b) culturing the host cell under conditions which allow proliferationof the host cell and expression and secretion of the target polypeptidefrom the host cell

(c) and recovering the target polypeptide.

According to a further aspect of the present invention, there isprovided a Chinese hamster ovary cell, preferably a CHOK1, DG44, DUXKB11or CHO pro3-cell, transfected with an expression vector comprising anexpression cassette comprising a polynucleotide encoding the targetpolypeptide operably linked to a fibronectin secretion leader sequenceor a functional equivalent thereof.

The target polypeptide encoded in the further aspect of the presentinvention is preferably comprises a monoclonal antibody. An expressioncassette comprising polynucleotides encoding both heavy and light chainsof a monoclonal antibody, preferably each operably linked to afibronectin secretion leader, may be employed. In some embodiments,separate expression cassettes comprising heavy and light chains areemployed, which may be located on separate vectors, but are oftenlocated on the same vector. The fibronectin secretion leaders employedmay be the same or different, but are preferably the same.

In many preferred embodiments, the expression cassette comprises ahousekeeping gene promoter, especially an hEF1a promoter operably linkedto the polynucleotide encoding the target polypeptide, and when two ormore expression cassettes are employed, each expression cassettecomprises a housekeeping gene promoter, preferably the same promoter,and most preferably an hEF1a promoter.

The, or each, expression cassette for the target polypeptide preferablycomprises a bovine growth hormone polyA sequence.

The expression vector preferably comprises a selection marker, mostpreferably a dihydrofolate reductase marker system. In certaininstances, the dihydrofolate reductase marker system comprises anexpression cassette further comprising a murine phosphoglycerate kinasepromoter.

A DNA construct comprising an expression cassette comprising a promotereffective in a mammalian cell and a polynucleotide encoding a targetpolypeptide operably linked to a fibronectin secretion leader sequenceforms another aspect of the present invention.

The DNA constructs preferably comprise separate expression cassettes forheavy and light chains of a monoclonal antibody. Most preferably, eachexpression cassette comprises the same promoter, especially ahousekeeping gene promoter, most especially an hEF1a promoter.Particularly preferably, each expression cassette further comprises abovine growth hormone polyA sequence. The DNA constructs oftenadvantageously comprise a selection marker, most preferably adihydrofolate reductase marker system. In certain instances, thedihydrofolate reductase marker system comprises an expression cassettefurther comprising a murine phosphoglycerate kinase promoter.

The present invention is illustrated without limitation by the followingexamples.

EXAMPLE 1

For each secretion leader (SL) being assessed, two single gene vectorswere constructed which contained either the hγ1 FL heavy chain (HC) ofan anti-MUC-1 MAb or the lambda light chain (LC) of an anti-MUC-1 MAb.Each expression cassette consisted of a rat CMV promoter, functionallylinked to a polynucleotide sequence encoding the secretion leader whichwas linked in frame to a polynucleotide sequence encoding the HC or LCmature polypeptide and a human betaglobin polyA sequence. The structureof the expression cassettes is illustrated in FIG. 1.

Secretion leaders employed were as follows:

Secretion leader A:  (SEQ ID No. 5)human collagen, sequence MLSFVDTRTLLLLAVTLCLATCQS Secretion leader B: (SEQ ID No. 1) human fibronectin, sequence MLRGPGPGLLLLAVQCLGTAV PSTGASecretion leader C:  (SEQ ID No. 2)Chinese hamster fibronectin, sequence MLRGPGPGLLL AVLCLGTAVRCTEA Secretion leader D:  (SEQ ID No. 6)Chinese hamster albumin, sequence MKWVTFLLLLFVSDS AFS 

CHO DG44 host cells were counted and seeded onto wells of a 6 well plateat 1.2×10⁶ cellswell in MEM-α medium supplemented with 10% serum, 2 mMGlutamine and 0.45% glucose, and incubated overnight at 36.5° C., 7.5%CO₂.

For each transfection 4 μg of the HC and LC single gene vectors (2 μg)were mixed together and diluted in 250 μL serum free MEM-α medium (LifeTechnologies). A mock transfection (PBS only) was also included. Foreach transfection 12.5 μL Lipofectamine 2000 (Life Technologies) wasdiluted in 250 μL serum free MEM-α medium and mixed. The mixture wasincubated at room temperature (15-25° C.) for 5 minutes. The diluted DNAand Lipofectamine 2000™ reagent were combined, mixed and incubated for20 minutes at room temperature. A further 500 μL MEM-α medium was addedto each transfection mix, growth medium was removed from the well andthe complex was then added to a well of the 6-well plate containing thecells. After 5 hours the medium was removed and fresh growth medium wasadded. Cells were incubated for 5 days at 36.5° C., 7.5% CO₂.Supernatant was harvested and clarified by centrifugation. Antibodytitre was determined using an Octet (Forte Bio) protein A assay.

The results are given in Table 1 below.

TABLE 1 Secretion leader used Mean antibody titre (mg/l) A 2.91 B 7.79 C8.85 D 1.89

The antibody produced is recovered from the supernatant by Protein Acapture, elution at low pH and purified by cation exchangechromatography followed by anion exchange chromatography. Eluent fromthe anion exchange chromatography is subject to viral nanofiltration,followed by buffer exchange and concentration.

EXAMPLE 2

Vector Construction

Double gene vectors were constructed which contained a hEF1a promoterdriving expression of both the hγ1 FL heavy chain of an anti-MUC-1 MAband the human lambda light chain of an anti-MUC-1 MAb.

Further double gene vectors were constructed where the hEF1α promoterwas exchanged for either a hCMV-MIE promoter or rat CMV promoter.

Each expression cassette within the double gene vectors consisted of thepromoter functionally linked to a polynucleotide sequence encoding theCHO fibronectin signal peptide of Example 1, which was linked in frameto a polynucleotide sequence encoding the HC or LC mature polypeptide.Correct mRNA processing was ensured by the presence of a bovine growthhormone poly A sequence.

To allow selection of stable cell lines the vectors also contained acopy of the mouse dyhydrofolate reductase (dhfr) gene under control ofthe murine phosphoglycerate (mPGK) promoter and the hygromycinresistance gene under the control of the thymidine kinase (TK) promoter.

Routine Subculture of CHO DG44 Cells:

CHO DG44 cells were routinely cultured in suspension shaker flasks inEX-CELL ACF CHO medium (Sigma) supplemented with 8 mM L-glutamine and 1×HT supplement (Life Technologies). Cells were seeded at a concentrationof 2×10⁵ cellsml, and cells were split every 3 days. Flasks werecultured at 37° C., 7.5% CO₂ in an orbital shaking incubator at 140 rpm.

Transfections for Generation of Stable Cell Lines

Cells used for transfections were grown in cell suspension culture, asdetailed above. Cells from growing cultures were centrifuged andre-suspended to a concentration of 2×10⁷ cellsmL. A 0.1 mL volume of thecell suspension and 4 μg of linearised plasmid DNA were added to anelectroporation cuvette. The cuvette was then placed in the Amaxanucleofector (Lonza) and nucleofected. Following transfection, the cellswere added to 20 ml pre-warmed EX-CELL ACF CHO medium (Sigma)supplemented with 8 mM Glutamine and 1×HT supplement in a T75 flask.Transfected cells were incubated at 37° C., 7.5% CO₂. Following theremoval of hypoxanthine and thymidine (HT) (48 hrs post transfection)from the medium and addition of 400 μgml Hygromycin B (Invitrogen) and25 nM MTX (144 hrs post transfection) cells were plated out into 96 wellplates at 5000 cellswell (2.5×10⁴mL). The plates were incubated at 37°C. in an atmosphere of 7.5% CO₂ in air. The plates were monitored forcolony growth up to approximately three weeks after transfection.Supernatant from up to 100 wells containing cell growth were harvestedand analysed for the Antibody using an Octet (Forte Bio) protein Aassay. The top 24 expressing colonies were expanded into 24 well platesand cultured for 10 days. Supernatant was then assayed for the Antibodyusing an Octet (Forte Bio) protein A assay. The results are given inTable 2 below.

TABLE 2 hEF1α hCMV-MIE Rat CMV 96 wp 24 wp 96 wp 24 wp 96 wp 24 wp MaxExp 7.3 18.0 6.1 3.2 6.1 nd Level (μg/mL) Mean Exp 3.3 4.2 0.7 1.3 0.6nd Level (μg/mL)

EXAMPLE 3

Purification of Antibody from CHO Supernatant

Supernatant from recombinant CHO DG44 cell lines generated using thehEF1α promoter double gene vector described in Example 2 was purifiedusing protein A resin. 350 mL of clarified harvest was loaded onto apre-packed column containing MabSelect SuRe resin (GE Healthcare). Resinwas washed first with 20 mM Sodium Phosphate, 1M NaCl (pH7.0) and thenwith 20 mM Sodium Phosphate (pH7.0). Antibody was then eluted with 100mM Acetic acid. Recovered product was quantified using an Octet (ForteBio) protein A assay and is shown in Table 3.

TABLE 3 Volume (mL) Concentration (mg/mL) Clarified Harvest 350 1.3Eluted Antibody 50.55 7.7

The invention claimed is:
 1. A Chinese hamster ovary cell transfectedwith an expression vector comprising an expression cassette comprising apolynucleotide encoding a heterologous target polypeptide operablylinked to a fibronectin secretion leader sequence having the sequence ofSEQ ID NO:
 2. 2. A Chinese hamster ovary cell according to claim 1,wherein the heterologous target polypeptide comprises a monoclonalantibody.
 3. A Chinese hamster ovary cell according to claim 2, whereinseparate expression cassettes comprising heavy and light chains areemployed.
 4. A Chinese hamster ovary cell according to claim 3, whereinthe expression cassettes are located on the same vector.
 5. A Chinesehamster ovary cell according to claim 3, wherein each expressioncassette comprises a hEF1a promoter operably linked to thepolynucleotide encoding the heterologous target polypeptide.
 6. AChinese hamster ovary cell according to claim 5, wherein each expressioncassette comprises a bovine growth hormone polyA sequence.
 7. A Chinesehamster ovary cell according to claim 6 which further comprises adihydrofolate reductase marker system.
 8. A Chinese hamster ovary cellaccording to claim 7, wherein the dihydrofolate reductase marker systemcomprises a murine phosphoglycerate kinase promoter.
 9. A process forthe production of a heterologous target polypeptide which comprises: (a)culturing the Chinese hamster ovary cell of claim 1 in a medium undercondition sufficient to express the heterologous target polypeptide; and(b) recovering the heterologous target polypeptide from the medium. 10.A process for the production of a heterologous target polypeptide whichcomprises: (a) preparing the Chinese hamster ovary cell of claim 1 bytransfection of a Chinese hamster ovary cell with an expression vectorfor expressing a heterologous target polypeptide in the Chinese hamsterovary cell, the expression vector comprising an expression cassettecomprising a polynucleotide encoding the heterologous target polypeptideoperably linked to a fibronectin secretion leader sequence having thesequence of SEQ ID NO: 2; (b) culturing the Chinese hamster ovary cellunder conditions which allow proliferation of the Chinese hamster ovarycell and expression and secretion of the heterologous target polypeptidefrom the Chinese hamster ovary cell; and (c) recovering the heterologoustarget polypeptide.
 11. A process according to claim 9, wherein theexpression cassette comprises an hEF1a promoter.
 12. A process accordingto claim 11, wherein the expression cassette comprises a polyA sequence.13. A process according to claim 12, wherein the poly A sequence isselected from human betaglobin polyA, bovine growth hormone polyA SV40early or late poly A sequences.
 14. A process according to claim 9,wherein two expression cassettes are employed, one expression cassettecomprising a polynucleotide encoding a light chain of a monoclonalantibody, and a second expression cassette comprising a polynucleotideencoding a heavy chain of a monoclonal antibody.
 15. A process accordingto claim 14, wherein the two expression cassettes comprise the samepromoter, secretion leader and polyA sequence.
 16. A process accordingto claim 15, wherein the promoter is an hEF1a promoter, and the polyAsequence is bovine betaglobin polyA sequence.
 17. A process for theproduction of a heterologous polypeptide, comprising culturing theChinese hamster ovary cell according to claim 1 thereby producing theheterologous polypeptide.